We are interested in the transcriptional control of T cell development and function. Specifically, we study the gene expression programs that control the choice by intrathymic T cell precursors of the CD4 or CD8 lineage, and perpetuate lineage differentiation in post-thymic T cells. T cells are essential for immune responses. Conventional'T cells recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins (called coreceptors) that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and strongly correlates with both MHC specificity and functional differentiation. That is, the general rule is that MHC I-specific T cells are CD4-CD8+ and cytotoxic (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- and helper or regulatory (CD4 cells). This double correspondence is essential to the proper function of the immune system and is established in the thymus, where CD4 and CD8 T cells emerge as separate lineages from precursors expressing both CD4 and CD8 (double positive, DP). Deciphering the transcriptional regulatory networks that decide and maintain CD4-CD8 lineage differentiation is the target of our current research. Two conceptually distinct processes are thought to be involved in the differentiation of CD4 cells: initiating expression of genes specific of the CD4 lineage, and repressing genes characteristic of the CD8 lineage. We previously identified the transcription factor Thpok, whose expression in T cells is specific of the CD4 lineage, as a major repressor of CD8-lineage gene expression and CD8 T cell differentiation. Furthermore, we had shown that another transcription factor, Gata3, was required for the expression of Thpok, and was therefore important to specify CD4-lineage gene expression. We had also found that these distinct functions of Thpok and Gata3 were conserved in a specific subset of non conventional T cells recognizing lipid antigens (invariant natural killer cells, iNK T cells). In the period covered by this report, we have addressed two important questions raised by these findings. First, what is the functional imprint of Gata3 in CD4-differentiating cells? To address this question, we have generated transgenic mice constitutively expressing Gata3 in developing thymocytes at the physiological levels normally seen in the direct precursors of CD4 T cells. Using this approach, we have observed that Gata3 is not sufficient to promote expression of CD4-lineage genes, including that of Thpok. In contrast, and unexpectedly, transgenic Gata3 impairs the expression of genes specific of CD8-lineage cells, including CD8 itself and the transcription factor Runx3. Despite this effect, expression of the Gata3 transgene in Thpok-deficient cells fails to restore their ability to become CD4 cells, consistent with our previous proposal that the two factors have non-redundant functions. The second question we have addressed is the identity of the factor(s) directing expression of CD4-lineage genes, a key unknown in the field. While Gata3 is a prime candidate for such a function, our observation that Gata3 is required but not sufficient for Thpok expression supports the idea that other factors are important for CD4-lineage gene expression. In addition, it has been proposed that Thpok proteins themselves directly promote expression of CD4-lineage genes, including that of the Thpok gene itself. To address these questions, we have undertaken large-scale analyses of gene expression and chromatin modifications in CD4 and CD8-differentiating cells. The first objective of these analyses, that utilize high-throughput deep-sequencing combined with bioinformatics, is to characterize gene expression and epigenetic patterns at distinct stages of lineage differentiation. The second objective is to analyze the contribution of Thpok to lineage differentiation by comparing the transcriptome and epigenome of wild-type and Thpok-deficient cells at similar developmental stages. Last, we intend to use this approach to discover new genes involved in specifying the CD4-lineage gene expression program. Although these experiments are still in progress, they have already reached a few firm conclusions. First, CD4-CD8 differentiation is accompanied by major epigenetic reconfiguration at lineage specific genes. In addition, the apparent symmetry between CD4 and CD8 lineage differentiation masks substantial differences in epigenetic modification patterns, notably at the loci encoding CD4 and CD8 genes. Interestingly, key hallmarks of CD4-lineage differentiation are established in a Thpok-independent manner, including epigenetic reconfiguration at the Thpok locus, supporting the idea that Thpok is not needed to specify expression of CD4-lineage genes. In contrast, the lack of Thpok results in the widespread opening of CD8-lineage gene loci in CD4-lineage precursors. We are currently pursuing these analyses focusing on the discovery of new transcripts potentially involved in the lineage differentiation process.